1. Field of the Invention
This invention relates to mutant angiogenin genes produced by site-specific mutagenesis and recombinant DNA techniques and includes DNA sequences for the mutant angiogenic genes which encode mutant proteins with increased angiogenic and ribonucleolytic activities. Additionally, the invention relates to methods of expression of mutant angiogenic proteins with increased angiogenic and ribonucleolytic activities as well as the resulting mutant angiogenic proteins.
It has now been unexpectedly found that replacement of the aspartic acid at or corresponding to position 116 (Asp-116) of human angiogenin with another amino acid, in particular, asparagine (Asn), alanine (Ala), or histidine (His), by site-specific mutagenesis of an angiogenin gene, results in a significant enhancement of both the angiogenin and ribonucleolytic activity of angiogenin.
2. Background of the Art
Angiogensis, the process of developing a hemovascular network, is essential for the growth of solid tumors and is a component of normal wound healing and growth processes. It has also been implicated in the pathophysiology of atherogenesis, arthritis, and diabetic retinopathy. It is characterized by the directed growth of new capillaries toward a specific stimulus. This growth, mediated by the migration of endothelial cells, may proceed independently of endothelial cell mitosis.
The molecular messengers responsible for the process of angiogenesis have long been sought. Greenblatt and Shubik (J. Natl. Cancer Inst. 41: 111-124, 1968) concluded that tumor-induced neovascularization is mediated by a diffusible substance. Subsequently, a variety of soluble mediators have been implicated in the induction of neovascularization. These include prostaglandins (Auerback, in Lymphokines, Pick and Landy, eds., 69-88, Academic Press, New York, 1981), human urokinase (Berman et al., Invest. Opthalm. Vis. Sci. 22; 191-199, 1982), copper (Raju et al., J. Natl. Cancer Inst. 69: 1183-1188, 1982), and various "angiogenesis factors."
Angiogenesis factors have been derived from tumor cells, wound fluid (Banda et al., Proc. Natl. Acad. Sci. USA 79: 7773-7777, 1982; Banda et al., U.S. Pat. No. 4,503,038), and retinal cells (D'Amore, Proc. Natl. Acad. Sci. USA 78: 3068-3072, 1981). Tumor-derived angiogenesis factors have in general been poorly characterized. Folkman et al. (J. Exp. Med. 133: 275-288, 1971) isolated tumor angiogenesis factor from the Walker 256 rat ascites tumor. The factor was mitogenic for capillary endothelial cells and was inactivated by ribonuclease (RNase). Tuan et al. (Biochemistry 12: 3159-3165, 1973) found mitogenic and angiogenic activity in the nonhistone proteins of the Walker 256 tumor. The active fraction was a mixture of proteins and carbohydrate. A variety of animal and human tumors have been shown to produce angiogenesis factor(s) (Phillips and Kuman, Int. J. Cancer 23: 82-88, 1979) but the chemical nature of the factor(s) was not determined. A low molecular weight non-protein component from Walker 256 tumors has been shown to be angiogenic and mitogenic (Weiss et al., Br. J. Cancer 40: 493-496, 1979). An angiogenesis factor with a molecular weight of 400-800 daltons was purified to homogeneity by Fenselau et al. (J. Biol. Chem. 256: 9605-9611, 1981), but it was not further characterized. Human lung tumor cells have been shown to secrete an angiogenesis factor comprising a high molecular weight carrier and a low molecular weight, possibly non-protein, active component (Kumar et al., Int. J. Cancer 32: 461-464, 1983). Vallee et al. (Experientia 41: 1-15, 1985) found angiogenic activity associated with three fractions from Walker 256 tumors. Tolbert et al. (U.S. Pat. No. 4,229,531) disclose the production of angiogenesis factor from the human adenocarcinoma cell line HT-29, but the material was only partially purified and was not chemically characterized. Isolation of genes responsible for the production of the above described angiogenesis factors has not been reported at least in part due to the lack of purity and characterization of the factors.
Isolation of angiogenesis factors has employed high performance liquid chromatography (Banda et al., ibid); solvent extraction (Folkman et al., ibid); chromatography on silica gel (Fenselau et al., ibid); DEAE cellulose (Weiss et al., ibid), or Sephadex Tuan et al, ibid); and affinity chromatography (Weiss et al., ibid).
Recently, Vallee et al. (U.S. Pat. No. 4,727,137 which is hereby incorporated by reference) have purified an angiogenic protein from a human adenocarcinoma cell line. The protein has been identified in normal human plasma (Shapiro, et al. Biochem. 26: 5141-5146, 1987). The purified protein, known as angiogenin, was chemically characterized and its amino acid sequence determined. Two distinct, although apparently linked, biological activities have been demonstrated for the human tumor-derived angiogenin. First, it was reported to behave as a very potent angiogenic factor in vivo (Fett et al., Biochem. 24: 5480-5456, 1985). Second, it has been found to exhibit a characteristic ribonucleolytic activity (Shapiro et al., Biochem. 25: 3527-3532, 1986).
In addition, Vallee et al. (U.S. Pat. No. 4,721,672, which is hereby incorporated by reference) recently have cloned the gene (both cDNA and genomic) encoding the angiogenic protein from the human adenocarcinoma cell line described and claimed in the above referenced U.S. Pat. No. 4,727,137. They have cloned the gene in vectors and have transformed or transfected host cells with recombinant vectors encoding the angiogenin gene. Such transformed or transfected cells express a human angiogenin protein.
Denefle et al. (Gene 56: 61-70, 1987), have prepared a synthetic gene coding for human angiogenin. The gene was designed to use codons found in highly expressed E. coli proteins and was ligated into a pBR322-derived expression vector constructed to contain the E. coli tryptophan (trp) promoter. This E. coli-produced angiogenin was found to be insoluble but could be easily renatured and purified. The purified angiogenin exhibited angiogenic activity and ribonucleolytic activity similar to that described for natural angiogenin purified by Vallee et al. (U.S. Pat. No. 4,727,137) from human adenocarcinoma cells.
Hoechst (German Patent Application P3716722.7) has prepared a different synthetic gene for angiogenin with a leucine at position 30 instead of the methionine at position 30 in the natural angiogenin gene described by Vallee et al. (U.S. Pat. No. 4,721,672). In addition, this synthetic gene was designed to use codons preferentially expressed in E. coli. The gene was subcloned into a vector containing a modified trp promoter (European Patent Application 0198415) and a translation initiation region (TIR) sequence (Gene 41: 201-206, 1986; EMBO J. 4: 519-526, 1985) to increase translation efficiency. The synthetic gene is under direct control of the trp promoter and expression is induced by addition of indole-3-acrylic acid or by tryptophan starvation. The leu-30 angiogenin protein could be purified and was found to exhibit angiogenic and ribonucleolytic activity similar to that of natural angiogenin.
All the angiogenin proteins just described, whether plasma-derived, tumor cell-derived or recombinant DNA-derived (cDNA, genomic DNA or synthetic gene derived) exhibit both angiogenic activity and ribonucleolytic activity. These two activities have not yet been separated. Indeed, one of the most intriguing features of angiogenin is its structural homology with mammalian pancreatic ribonucleases (RNases). Overall, there is a 35% sequence identity between human pancreatic RNase and angiogenin (Strydom et al., Biochemistry 24: 5486-5494, 1985). This structural relationship should permit the study of the mechanism of action of angiogenin, as well as the relationship between the angiogenic and enzymatic (ribonucleolytic) activities of angiogenin.
Because angiogenesis factors play an important role in wound healing (Rettura et al. FASEB Abstract #4309, 61st Annual Meeting, Chicago, 1977) and may find applicability in the development of screening tests for malignancies (Klagsburn et al., Cancer Res. 36: 110-114, 1976; Brem et al. Science 195: 880-881, 1977), it is clearly advantageous to produce angiogenic proteins in sufficient quantities to permit their application in therapy and diagonsis. The techniques of genetic engineering are ideally suited to increase production levels of these proteins. The cloning of genes encoding angiogenic proteins, such as described in U.S. Pat. No. 4,721,672, is a necessary first step in such a large-scale production. In addition to increasing production levels of angiogenic proteins, it would be highly advantageous to use cloned genes to produce mutant or variant angiogenic proteins with angiogenic activity that is much increased over wild-type activity. The techniques of site-specific mutagenesis and genetic engineering are ideally suited to producing proteins with such increased activity. Although it is clear that the amino acids of an angiogenic protein may be modified by such techniques to produce proteins with altered biological activities, it is difficult to predict which amino acids should be altered and whether such an alteration will increase or decrease biological activity. U.S. Pat. No. 4,721,672 states that the cysteines at positions 26, 39, 57, 81, 92 and 107, and histidines at positions 13 and 114, and the lysine at position 40 should be preferred sites for replacement by other amino acids using site-specific mutagenesis.
Furthermore, it may in some instances be desirable to obtain these mutant angiogenic proteins with increased angiogenic activity from non-tumor cells, such as in the case of human therapeutics, where contamination with certain tumor products would be unacceptable and where an increase in biological activity could permit the use of lower dosage levels. This invention therefore provides for the production of mutant angiogenic proteins in non-tumor cells with increased angiogenic activity using site-specific mutagenesis and recombinant DNA techniques.